The present invention relates to the use of specific transporter cargo conjugate molecules for the transport of a substance of interest (cargo molecule) into white blood cells. Said transporter cargo conjugate molecules may be used for the treatment, prophylaxis, attenuation and/or amelioration of a disease and/or disorder involving white blood cells. The present invention also relates to manufacture of said transporter cargo conjugate molecules, to a method of transporting a substance of interest (cargo) into a white blood cell and to a white blood cell comprising said transporter cargo conjugate molecules or fragments thereof.
Techniques enabling efficient transfer of a substance of interest from the external medium into tissue or cells, and particularly to cellular nuclei, such as nucleic acids, proteins or cytotoxic agents, but also of other compounds, are of considerable interest in the field of biotechnology. These techniques may be suitable for transport and translation of nucleic acids into cells in vitro and in vivo and thus for protein or (poly-)peptide production, for regulation of gene expression, for induction of cytotoxic or apoptotic effects, for analysis of intracellular processes and for the analysis of the effects caused by the transport of a variety of different cargos into a cell (or cell nucleus), etc.
One important application of such a transfer of a cargo of interest from the external medium into tissue or cells is targeted drug delivery. Targeted drug delivery relates to the tissue-/cell specific delivery of a pharmaceutically active drug. The intention is to achieve after (systemic) administration over time a high concentration of the pharmaceutically active drug at or in the cells and tissues of interest while at the same time the concentration of the pharmaceutically active drug in the remaining cells and tissues is kept at a low level. This improves efficacy of the therapy and reduces side effects. Such targeted drug delivery can be mediated for example by highly specific antibodies.
A further important application of such a transfer of a cargo of interest from the external medium into tissue or cells is gene therapy, wherein the cargo is typically a nucleic acid or a gene. Although this technique has shown some rather promising developments in the last decades, gene transfer is typically limited by the inability of the gene transfer vectors to effectively transfer the biologically active cargo into the cytoplasm or nuclei of cells in the host to be treated without affecting the host genome or altering the biological properties of the active cargo.
In this respect, several techniques have been developed in an effort to more efficiently transfect e.g. nucleic acids, such as DNA or RNA, into cells. Transfection of nucleic acids into cells or tissues of patients by methods of gene transfer is a central method of molecular medicine and plays a critical role in therapy and prevention of numerous diseases.
Representative examples of gene transfer methods include general (physical or physico-chemical) methods such as coprecipitating nucleic acids with calcium phosphate or DEAE-dextran, a method which enables nucleic acids to penetrate the plasma membrane and then enter the cell and/or nucleus. However, this technique suffers from low transfer efficiency and a high percentage of cell death. Additionally, this method is restricted to in vitro or ex vivo methods, but is not applicable to in vivo situations due to its very nature.
The same holds true for methods involving in vitro electroporation. In vitro electroporation is based on the use of high-voltage current to make cell membranes permeable to allow the introduction of new nucleic acids, e.g. DNA or RNA, into the cell. However, such methods are typically not suitable in vivo. Furthermore, this technique also suffers from low transfer efficiency and a high percentage of cell death.
Further well known physical or physico-chemical methods include (direct) injection of (naked) nucleic acids or biolistic gene transfer. Biolistic gene transfer (also known as biolistic particle bombardment) is a method developed at Cornell University that allows introducing genetic material into tissues or culture cells. Biolistic gene transfer is typically accomplished by surface coating metal particles, such as gold or silver particles, and shooting these metal particles, comprising the adsorbed DNA, into cells by using a gene gun. Similar as discussed above this method is restricted to in vitro or ex vivo methods, but is usually not applicable in in vivo situations.
Other methods utilize the transport capabilities of so called transporter molecules. Transporter molecules to be used in this context typically may be divided into viral vectors on the one hand, i.e. transporter molecules, which involve viral elements, and nonviral vectors on the other hand.
The most successful gene therapy strategies available today rely on viral vectors, such as adenoviruses, adeno-associated viruses, retroviruses, and herpes viruses. These viral vectors typically employ a conjugate of a virus-related substance with a strong affinity for DNA and a nucleic acid. Due to their infection properties, viruses or viral vectors have a very high transfection rate. The viral vectors typically used are genetically modified in a way that no functional infectious particles are formed in the transfected cell. In spite of this safety precaution, however, there are many problems associated with viral vectors related to immunogenicity, cytotoxicity, and insertional mutagenesis. As an example, the risk of uncontrolled propagation of the introduced therapeutically active genes or viral genes cannot be ruled out, e.g., because of possible recombination events. Additionally, the viral conjugates are difficult to use and typically require a long preparation prior to treatment (see, e. g., U.S. Pat. No. 5,521,291).
Nonviral vectors are not as efficient as viral vectors if it comes to gene therapy; however, many of them have been developed to provide a safer alternative in gene therapy. Some of the most common nonviral vectors include polyethylenimine, dendrimers, chitosan, polylysine, and (poly-)peptide based transporter systems, e.g. many types of (poly-)peptides, which are generally cationic in nature and able to interact with nucleic acids such as plasmid DNA through electrostatic interactions. Additionally, nonviral vectors allow also for delivery of drugs not based on nucleic acids.
For successful delivery, the nonviral vectors, in particular (poly-)peptide based transporter systems, must be able to overcome many barriers. Such barriers include protection of the cargo moiety, e.g. of DNA or other compounds, during transport and prevention of an early degradation or metabolisation of the cargo moiety in vivo. In case of nucleic acids, such as DNA and RNA molecules, the nonviral vectors must furthermore be capable to specifically deliver these molecules for efficient gene expression in target cells.
Especially with respect to nucleic acids such as DNA and RNA molecules there are presently 4 barriers nonviral vectors must overcome to achieve successful gene delivery (see e.g. Martin et al., The AAPS Journal 2007; 9 (1) Article 3). The nonviral vector must be able to 1) tightly compact and protect the nucleic acids, 2) it must able to target specific cell-surface receptors, 3) the nonviral vector must be capable to disrupt the endosomal membrane, and 4) it has to deliver the nucleic acid cargo to the nucleus and allow translation of an encoded protein or (poly-)peptide sequence.
Such nonviral vectors, particularly (poly-)peptide-based nonviral vectors, are advantageous over other nonviral strategies in that they are in general able to achieve all 4 of these goals, however, with different efficiency regarding the different barriers.
As an example, cationic (poly-)peptides rich in basic residues such as lysine and/or arginine are able to efficiently condense nucleic acids such as DNA into small, compact particles that can be stabilized in serum. Furthermore, attachment of a (poly-)peptide ligand to the polyplex allows targeting to specific receptors and/or specific cell types. Polyplexes or cationic polymers as mentioned above typically form a complex with negatively charged nucleic acids leading to a condensation of nucleic acids and protecting these nucleic acids against degradation. Transport into cells using polyplexes (cationic polymers) typically occurs via receptor mediated endocytosis. Thereby, the DNA is coupled to a distinct molecule, such as Transferrin, via e.g. the polyplex poly-L-lysine (PLL), which binds to a surface receptor and triggers endocytosis. Polyplexes (cationic polymers) include e.g. poly-L-lysine (PLL), chitosan, polyethylenimine (PEI), polydimethylaminoethylmethacrylate (PD-MAEMA), polyamidoamine (PAMAM). Such effects are also known from nanoplexes (nanoparticular systems) or lipoplexes (liposomal systems). Nanoplexes (nanoparticular systems) typically involve the use of polyacrylates, polyamides, polystyrene, cyanoacrylates, polylactat (PLA), poly(lactic-co-glycolic acid) (PLGA), etc. Lipoplexes or liposomal systems typically involve the use of cationic lipids, which are capable to mimic a cell membrane. Thereby, the positively charged moiety of the lipid interacts with the negatively charged moiety of the nucleic acid and thus enables fusion with the cell membrane. Lipoplexes or liposomal systems include, e.g. DOTMA, DOPE, DOSPA, DOTAP, DC-Chol, EDMPC, etc.
In this context, receptor-mediated endocytosis is also widely exploited in experimental systems for the targeted delivery of cargos such as nucleic acids or therapeutic agents into cells. During receptor-mediated endocytosis the cargo-containing complexes are either selectively internalized by receptors located in the cell membrane which are specific for the cargos, or by specific antibodies located in membrane constituents. Endocytotic activity has been described for many receptors including IgG Fc, somatostatin, insulin, IGF-I and -II, transferrin, EGF, GLP-1, VLDL or integrin receptors, etc.
Different (poly-)peptide or protein sequences have been tested widely for their use in gene transfer methods via receptor-mediated endocytosis. Interestingly, the isolation of (poly-)peptide sequences that direct efficient receptor-mediated endocytosis have been profoundly boosted by the use of phage display technologies. Phage display libraries are extremely powerful tools that provide for an almost unlimited source of molecular variants including modifications of natural ligands or cargo moieties to cell receptors and short (poly-)peptides. Similar libraries have also been injected directly into mice and (poly-)peptide sequences have been successfully isolated that show a 13-fold selectivity for brain and kidney.
Proprotein convertases may serve as an example of (poly-)peptide or protein sequences that may be used for transport of molecules into cells. Proprotein convertases are an example of a cell surface receptor which gets internalized through receptor mediated endocytosis. These proteins have been shown to be responsible for conversion of precursors of (poly-)peptide hormones, neuropeptides, and many other proteins into their biologically active forms. All cleavage sites for the proprotein convertase family obey to the consensus R—X—X—R. The mammalian proprotein convertases can be classified into three groups on the basis of their tissue distribution. Furin, PACE4, PC5/PC6, and LPCIPC7/PC8/SPC7 are expressed in a broad range of tissues and cell lines. In contrast, expression of PC2 and PC1/PC3 is limited to neuroendocrine tissues, such as pancreatic islets, pituitary, adrenal medulla and many brain areas. Expression of PC4 is highly restricted to testicular spermatogenic cells. The neuroendocrine-specific convertases, PC2 and PC1/PC3, are mainly localized in secretory granules. PC5/PC6A has also been reported to be localized to secretory granules. Furthermore, indirect evidence has suggested that a proportion of proprotein convertases molecules is present on the cell surface, and it has been shown that furin cycles between the TGN and the cell surface. Taken together, these properties indicate that proprotein convertases transport extracellular ligands into the intracellular space.
Advantageous are also so called translocatory proteins or protein transduction domains (PTDs). (Poly-)peptide sequences derived from translocatory proteins or protein transduction domains (PTDs) are typically able to selectively lyse the endosomal membrane in its acidic environment leading to cytoplasmic release of the polyplex. Translocatory proteins are considered as a group of (poly-)peptides capable of effecting transport of macromolecules between cells (translocatory proteins), such as HIV-1 TAT (HIV), antennapedia (Drosophila antennapedia), HSV VP22 (Herpes simplex), FGF or lactoferrin, etc. In contrast, protein transduction domains (PTDs) are considered as a group of (poly-)peptides capable of directing proteins and (poly-)peptides covalently bound to these sequences into a cell via the cell membrane (Leifert and Whitton: Translocatory proteins and protein transduction domains: a critical analysis of their biological effects and the underlying mechanisms. Molecular Therapy Vol. 8 No. 1 2003). Common to translocatory proteins as well as to PTDs is a basic region, which is regarded as mainly responsible for transport of the fusion (poly-)peptides since it is capable of binding polyanions such as nucleic acids. Without being bound thereto, PTDs may act similar to cationic transfection reagents using receptor dependent non-saturatable adsorptive endocytosis. PTDs are typically coupled to proteins or (poly-)peptides in order to effect or enhance a CTL response when administering a (poly-)peptide based vaccine (see review: Melikov and Chernomordik, Arginine-rich cell penetrating (poly-)peptides: from endosomal uptake to nuclear delivery, Cell. Mol. Life Sci. 2005).
Although there are several techniques known in the art which enable transfer of a substance of interest from the external medium into tissue or cells in general, there is still a great need for techniques allowing for efficient transfer into specific tissue and cell types.